The present invention is generally in the field of detecting analytes in a medium and, more particularly, the present invention relates to micro-contact printing of antibody-binding proteins onto a substrate for the development of single use, disposable sensors to indicate the presence of the analyte in a medium.
There are many systems and devices available for detecting a wide variety of analytes in various media. Most of these systems and devices are relatively expensive and require a trained technician to perform the test. There are many cases where it would be advantageous to be able to rapidly and inexpensively determine if an analyte were present. What is needed is a biosensor system that is easy and inexpensive to manufacture and is capable of reliable and sensitive detection of analytes, including smaller analytes. Additionally, what is needed is an easy flexible method of preparation of the biosensors which would permit optimum scale-up processing.
Sandstrom et al., 24 Applied Optics 472, 1985, describe use of an optical substrate of silicon with a layer of silicon monoxide and a layer of silicon formed as dielectric films. They indicate that a change in film thickness changes the properties of the optical substrate to produce different colors related to the thickness of the film. The thickness of the film is related to the color observed and a film provided on top of an optical substrate may produce a visible color change. The authors indicate that a mathematical model can be used to quantitate the color change, and that xe2x80x9c[c]alculations performed using the computer model show that very little can be gained in optical performance from using a multilayer structure . . . but a biolayer on the surface changes the reflection of such structures very little since the optical properties are determined mainly by the interfaces inside the multilayer structure. The most sensitive system for detection of biolayers is a single layer coating, while in most other applications performance can be by additional dielectric layers.xe2x80x9d
Sandstrom et al., go on to indicate that slides formed from metal oxides on metal have certain drawbacks, and that the presence of metal ions can also be harmful in many biochemical applications. They indicate that the ideal top dielectric film is a 2-3 nm thickness of silicon dioxide which is formed spontaneously when silicon monoxide layer is deposited in ambient atmosphere, and that a 70-95 nm layer silicon dioxide on a 40-60 nm layer of silicon monoxide can be used on a glass or plastic substrate. They also describe formation of a wedge of silicon monoxide by selective etching of the silicon monoxide, treatment of the silicon dioxide surface with dichlorodimethylsilane, and application of a biolayer of antigen and antibody. From this wedge construction they were able to determine film thickness with an ellipsometer, and note that the xe2x80x9cmaximum contrast was found in the region about 65 nm where the interference color changed from purple to blue.xe2x80x9d They indicate that the sensitivity of such a system is high enough for the detection of protein antigen by immobilized antibodies. They conclude xe2x80x9cthe designs given are sensitive enough for a wide range of applications. The materials, i.e., glass, silicon, and silicon oxides, are chemically inert and do not affect the biochemical reaction studied. Using the computations above it is possible to design slides that are optimized for different applications. The slides can be manufactured and their quality ensured by industrial methods, and two designs are now commercially available.
U.S. Pat. No. 5,512,131 issued to Kumar et al. describes a device that includes a polymer substrate having a metal coating. An antibody-binding protein layer is stamped on the coated substrate. The device is used in a process for stamping or as a switch. A diffraction pattern is generated when an analyte binds to the device. A visualization device, such as a spectrometer, is then used to determine the presence of the diffraction pattern.
However, the device described by Kumar et al. has several disadvantages. One disadvantage is that an extra visualization device is needed to view any diffraction pattern. By requiring a visualization device, the Kumar et al. device does not allow a large number of samples to be tested since it is not possible to determine the presence of an analyte by using the unaided eye.
U.S. Pat. No. 5,482,830 to Bogart, et al., describes a device that includes a substrate which has an optically active surface exhibiting a first color in response to light impinging thereon. This first color is defined as a spectral distribution of the emanating light. The substrate also exhibits a second color which is different from the first color (by having a combination of wavelengths of light which differ from that combination present in the first color, or having a different spectral distribution, or by having an intensity of one or more of those wavelengths different from those present in the first color). The second color is exhibited in response to the same light when the analyte is present on the surface. The change from one color to another can be measured either by use of an instrument, or by eye. Such sensitive detection is an advance over the devices described by Sandstrom and Nygren, supra, and allow use of the devices in commercially viable and competitive manner.
However, the method and device described in the Bogart, et al. patent has several disadvantages. One disadvantage is the high cost of the device. Another problem with the device is the difficulty in controlling the various layers that are placed on the wafer so that one obtains a reliable reading. What is needed is a biosensor device that is easy and inexpensive to manufacture and is capable of reliable and sensitive detection of the analyte to be detected.
The present invention provides an inexpensive and sensitive device and method for detecting analytes present in a medium. The device comprises a biosensing device having a substrate, preferably a metalized polymer film, upon which is printed a specific predetermined pattern of antibody-binding proteins such as Protein A or Protein G. Subsequent exposure to the antibody specific for the desired analyte results in patterned deposition of this antibody. Overall, this allows a modular production format such that large rolls of patterned protein may be made for use with different analytes. Then as needed, the final product may be made by exposure to the necessary antibody.
Upon attachment of a target analyte, which is capable of scattering light, to select areas of the polymer film upon which the protein and antibody are patterned, diffraction of transmitted and/or reflected light occurs via the physical dimensions and defined, precise placement of the analyte. A diffraction image is produced which can be easily seen with the eye or, optionally, with a sensing device.
The present invention utilizes methods of contact printing of patterned, antibody-binding proteins. These proteins bind to antibodies to pattern them on the surface as well as maintain the optimum orientation for the receptor antibodies. The receptor antibodies are specific for a particular analyte or class of analyte, depending upon the protein used. Methods of contact printing which would be useful in generating the sensing devices used in the present system are disclosed fully in U.S. patent application Ser. Nos. 08/707,456, now U.S. Pat. No. 6,020,047 and 08/769,594, now U.S. Pat. No. 6,048,623, both of which are incorporated herein by reference in their entirety. However, since these methods relate to self-assembling monolayers, the methods need to be altered slightly, as discussed below, to print the antibody-binding protein material as this material is not self-assembling.
Patterned antibody-binding protein layers with bound antibodies cause patterned placement or binding of analytes thereon. The biosensing devices of the present invention produced thereby may be used in one of two ways, depending on the size of the analyte. For analytes which are capable of causing diffraction by themselves, such as microorganisms, the system is used by first exposing the biosensing device to a medium that contains the analyte of choice and then, after an appropriate incubation period, transmitting a light, such as a laser, through the film or reflecting it off of the film. If the analyte is present in the medium and is bound to the antibodies on the patterned antibody-binding protein layer, the light is diffracted in such a way as to produce a visible image.
Optionally, for very small analytes such as proteins, the system may utilize xe2x80x9cdiffraction enhancing elementsxe2x80x9d which are capable of binding to the target analyte and to the biosensor and are capable of producing a substantial change in the height and/or refractive index, thereby increasing the diffraction efficiency of the biosensor and permitting the detection of smaller analytes. In use, a target analyte attaches either to the diffraction enhancing element, which then attaches to the biosensor, or directly to select areas of the polymer film upon which the protein and antibody are printed. Then diffraction of transmitted and/or reflected light occurs via the physical dimensions and defined, precise placement of the analyte. A diffraction image is produced which can be easily seen with the eye or, optionally, with a sensing device.
Another option for use of this sensor involves the detection of analytes which are antibodies. The sensing device could comprise only the patterned antibody-binding proteins, and then would be exposed to the medium plus diffraction enhancing particles which have an antibody specific to the antibody to be detected. The selection of the antibody on the particle is preferably made so that it does not bind nonspecifically to the patterned antibody-binding protein, but instead binds only when the analyte antibody is also bound. In this way, the diffraction enhancing elements would cause a substantial change in the height and/or refractive index if the analyte antibody is present, thereby causing a diffraction image to form. It is envisioned that the same format could be used with other immunoassay formats, such as lateral flow assays or microwell plates.xe2x80x9d
In other words, the antibody-binding protein and antibody layers with the analyte bound thereto can produce optical diffraction patterns to indicate the presence of the analyte. The light can be in the visible spectrum, and be either reflected from the film, or transmitted through it, and the analyte can be any compound or particle reacting with the antibody-binding protein layer. The light can be a white light or monochromatic electromagnetic radiation in the visible region. The present invention also provides a flexible support for an antibody-binding protein layer on gold or other suitable metal or metal alloy.
The present invention provides a low-cost, disposable biosensor which can be mass produced. It includes the use of surfaces patterned with an antibody-binding protein. Typically, these proteins bind an antibody by its constant region (Fc) so that the antibody""s antigen-binding regions (Fab) are free for optimum binding activity. The preparation of the patterned protein surfaces also allows maximum flexibility in the sensor production. The final step in production, capturing the desired antibody in the patterned areas, may be done as needed for the desired analyte (i.e. at the time of manufacture).
The biosensors of the present invention can be produced as a single test for detecting an analyte or it can be formatted as a multiple test device. The biosensors of the present invention can be used to detect contamination in garments, such as diapers, and to detect contamination by microorganisms.
The present invention can also be used on contact lenses, eyeglasses, window panes, pharmaceutical vials, solvent containers, water bottles, adhesive bandages, and the like to detect contamination.
These and other features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiments.